Field of the Invention
The invention relates to a multimeric non-coding nucleic acid molecule for modulating the activity of the human or animal immune system, a method of preparing same, and a vaccine which contains the multimeric non-coding nucleic acid molecule.
Brief Description of the Related Art
While adaptive immune response following selection of lymphocytes specific for the respective pathogen and clonal expansion and differentiation thereof into effector cells takes effect only in a delayed fashion (3-5 days), but then offers long-lasting protection from the respective pathogen through formation of an “immunologic memory”, cells of the innate immune system recognize pathogens on the basis of preserved pathogen-associated molecular patterns (PAMPs) with germ cell-encoded receptors and respond immediately. The reactions that are different for different types of cells include secretion of cytokines (e.g. IL-1, IL-6, TNF-α) and chemokines (e.g. IL-8/CXCL8, MIP-1α/β, MCP-1), activation of effector mechanisms (phagocytosis, respiratory discharge, liberation of bactericidal substances or lytic granula), expression of co-stimulatory molecules (CD80, CD86) as well as enhanced expression of MHC molecules. On the one hand, this recruits and activates effector cells capable of eliminating the invaded pathogen and, on the other hand, the cells of the adaptive immune system receive signals necessary for activation thereof.
To produce an improved immune response, CpG oligonucleotides (CpG ODN) have been used as a new class of immunomodulatory molecules. Such non-methylated CG motifs occur in bacterial DNA and represent a “danger signal” to the immune system. Being “pathogen-associated molecular patterns” (PAMPs), they mainly cause non-specific activation of the innate immune system (Krieg, Nat. Med 2003, 9: 831-835).
CpG ODNs induce a TH1-accentuated immune response via the cytokines interleukin-12, interferon-γ and tumor necrosis factor α.
Immunostimulatory nucleic acid sequences (ISS) bearing the above-mentioned CpG ODNs are only a few bases in length and do not function via expression of proteins encoded thereon.
The ISS are covalently closed nucleic acid molecules. They consist of oligonucleotides, whose bases can undergo partial pairing with themselves, and one or two loops which comprise 30 bases and several CG motifs and will be referred to as carrier molecules hereinbelow.
The strong stimulation of cellular immune response allows exertion of influence on regulatory cycles which, without intervention, would result in an immune activity unsatisfactory to the patient.
Modification of CpG ODNs with phosphorothioate backbone, as used in stabilization of “CpG DNA”, has a number of serious drawbacks, including in particular the toxicity being observed [Heikenwalder 2004, Levin 1999], as well as non-specific binding to proteins [Brown 1994].
For this reason, a new class of covalently closed immunostimulatory DNA has been developed (EP 1196178). These DNA molecules consist of two chemically synthesized DNA molecules having a self-complementary region at the 5′ and 3′ ends with palindromic overlaps so that ligation of the two DNA molecules produces a covalently closed molecule. These DNA molecules with CG motifs in the non-complementary region show similar activity as CpG ODN (enhanced expression of the surface molecules CD80, CD40, MHC on B cells and secretion of IL-6, IFN-γ, IFN-α, IL-12, TNF-α by PBMC), but, compared to CpG ODN with phosphorothioate backbone, have a somewhat different expression pattern and significantly lower toxicity in mice. However, this immunostimulatory DNA of the prior art has a number of drawbacks regarding the modulation of human or animal immune system activity. It is not always possible to modulate, especially trigger, the activity of the human or animal immune system at a desired level.